Functional Characterisation of Putative Mechanoreceptive Neurones in the Colorectum

Conscious control of defecation is an important physiological function for most animals; conveying survival benefits in predation and is essential for territorial marking. Effective sensing of bowel (colorectal) fullness/urge is required to consciously inhibit reflex defecation until it is beneficial to survival, or as in humans, it is socially acceptable. Mechanical distension of the colorectum by passage of faecal matter is transduced by sensory neurones. In ageing, bowel function is impaired with increased rates of constipation and incontinence, concurrent with reduced colorectal sensitivity. Multiple types of sensory neurone innervate the colorectum, but it is unclear which of these populations is responsible for low-threshold (i.e. not painful) mechanosensation linked to sensing fullness and urge to defecate, and how their sensitivity changes in age. Using single-cell RNA-sequencing, we have identified two populations of sensory neurones innervating the colorectum, one expressing the mechanosensitive ion channel Piezo2 at high levels. We hypothesise that the Piezo2-positive (Piezo2+) population are key low-threshold mechanosensors in health and that their function declines with age resulting in impaired defecation.

We will use a combination of genetics, physiology and behaviour to determine the contribution of Piezo2 and Piezo2+ neurones to colorectal mechanosensitivity and defecation in age. By using mice lacking Piezo2 specifically in sensory neurones (Piezo2CKO), we will ascertain the role of this ion channel in mechanosensitivity of the colorectum at ages up to 24 months. In addition, mice in which Piezo2+ neurones express GFP/Cre-recombinase (Piezo2GFP) will be used at comparable ages to selectively visualise, modulate and ablate this population of neurones. We hypothesise that wild-type mice will show decreased stool frequency and increased bead expulsion latencies in age, which will both be exacerbated in Piezo2CKO mice due to diminished colorectal mechanosensitivity in this critical neuronal subset. To directly measure colorectal mechanosensitivity, we will use an ex vivo colon-nerve preparation and make electrophysiological recordings in response to colorectal distension and probe/stretch stimuli. To substantiate a role for Piezo2+ neurones, similar experiments will be conducted in Piezo2GFP mice after selective neuronal ablation by colorectal injection of Cre-dependent diphtheria toxin A. To determine Piezo2+ neuronal loss during ageing, we will retrograde label colorectal sensory neurones in Piezo2GFP mice.

To fully address how Piezo2+ colorectal sensory neurones contribute to colorectal sensitivity and defecation we will additionally use chemo- and optogenetic methods. Firstly, using Cre-dependent Designer Receptor Exclusively Activated by Designer Drug hM3Dq, we will selectively excite Piezo2+ colorectal neurones in Piezo2GFP mice by injection of the hM3Dq agonist clozapine-N-oxide. By combining behaviours (stool frequency/bead expulsion latency) with measurements of colorectal sensitivity both in vivo (via visceromotor reflex (VMR) to colorectal distension) and ex vivo (colon-nerve electrophysiology), we will investigate how activation of these colorectal sensory pathways impacts defecatory function. Lastly, Piezo2GFP mice will be crossed with mice expressing Cre-dependent channel rhodopsin and a fibre optic cable implanted into the lumbosacral spinal column of the offspring enabling the selective activation of colorectal sensory neurones in freely moving animals. We will determine how this activation modulates stool frequency, bead expulsion latency and VMR.

In summary, we will determine how Piezo2 and Piezo2+ neurones subserve low-threshold colorectal mechanosensitivity and contribute to defecation. Importantly, this study will reveal how changes in Piezo2+ neuronal function contribute to ageing-related decline in colorectal mechanosensitivity that may underpin impaired bowel function in geriatric

Effective colorectal mechanosensation is a prerequisite for controlled defecation. Impaired colorectal mechanosensation occurs in ageing and may contribute to increased gastrointestinal dysfunction (constipation and incontinence) observed in geriatrics. Preliminary single cell RNA-seq data from retrograde labelled colorectal sensory neurones has identified a population innervating the colorectum expressing high levels of the mechanosensitive ion channel Piezo2. We hypothesise that Piezo2-positive sensory neurones are low-threshold colorectal mechanosensors under healthy conditions, play an important role in defecation and that their function declines with ageing.

We will use behaviour and electrophysiology to measure stool frequency, defecatory function and colorectal mechanosensitivity during ageing in mice lacking Piezo2 in sensory neurones (Piezo2CKO). Further experiments will examine how loss of Piezo2-positive neurones, or their specific excitation, impacts defecation, defecatory function and mechanosensitivity of colorectal sensory neurones. Ablation and excitation of Piezo2-positive neurones will be achieved by using mice with Piezo2-dependent Cre recombinase expression combined with either colorectal injection of adeno-associated viruses encoding diphtheria toxin A (DTA) or the Designer Receptor Exclusively Activated by Designer Drug (DREADD) hM3Dq respectively. We will also measure the visceromotor reflex (VMR) to colorectal distension (CRD) in wildtype and Piezo2CKO mice, as well as those in which DTA and hM3Dq have been injected to assess how Piezo2 and Piezo2-positive neurones influence colorectal sensitivity in vivo. Lastly, Piezo2GFP mice will be crossed with mice expressing Cre-dependent channel rhodopsin, a fibre optic ferrule implanted into the lumbosacral spinal column of offspring, and light pulses applied in freely moving animals to determine how activation of colorectal sensory neurones modulates defecatory function.

Who might benefit from this research?
This research programme is fundamental research that will elucidate the role of Piezo2 and Piezo2-positive nerve fibres to colorectal mechanosensitivity and the role that they play in the ageing associated decline in colorectal function. Therefore, the primary impact will be on those interested in gastrointestinal function and the neuroscience of ageing. However, results from our study also have the potential to highlight potential therapeutic strategies for treating age-related colorectal dysfunction, for example if Piezo2-positive fibres are identified as being the critical colorectal low-threshold mechanosensors and that their function declines with ageing then it would perhaps highlight the need for development of positive modulators of Piezo2.

The general public is also curious about science and the way in which the nervous system works to enable control of process in the body that we take for granted. Both the Researcher Co-investigator employed on the grant, Dr Hockley, and myself have a strong record of public engagement and this is something that we will build on for the duration of this grant, giving talks accompanied by a video made specifically for this grant (see Pathways to Impact for details) to GCSE/A-level students in local schools and Life Long Learning courses, as well as contributing to events such as the Cambridge Science Festival. Lastly, staff working on the project and in the research group would also benefit, both with regard to acquisition of new technical skills and through development of teaching and presentation skills.

How might they benefit from this research?
The work set out in this research programme will help establish the fundamental mechanisms of colorectal mechanosensation, how this relates to defecation, and how ageing influences these processes. Consequently, this work has the potential to not only influence other academics across a broad range of fields, but could also help to direct therapeutic avenues of research with regard to treatment of geriatric-related gastrointestinal medical conditions and possibly also providing input to the study of pain-related conditions, such as irritable bowel syndrome.

In addition to academic benefit and the long-term, potential therapeutic development, the general public will benefit in a more acute fashion: we will produce a short film entitled Losing Control: Understanding Your Gut in Age and Disease that will discuss the role of the nervous system in the gastrointestinal tract, as well as diseases of the gut and how these can be treated. The film would be made in collaboration with LittleDragonFilms, with whom I have prior experience, and would be hosted on the lab website, as well as on the Departmental Outreach page and circulated via personal and institutional Twitter/Facebook feeds, such as that run by Cambridge Neuroscience, as well as being used as part of outreach talks. Importantly, there would be a multiple-choice test (hosted by SurveyMonkey) that would measure the film's impact on learning and understanding.

Staff working on the project will develop a wide-range of technical skills, as reflected by the variety of techniques to be employed in the project. Moreover, there will be opportunities for research staff to present their work at conferences, thus developing presentation and networking skills. The Department of Pharmacology offers the opportunity for research staff to teach in practicals and small group tutorials, which in combination with partaking in Teaching and Learning Workshops organised by the School of Life Sciences, will provide an opportunity to develop a further skill-set. Staff will also be encouraged to take training courses offered by the Public Engagement Department and Researcher Development Programme to develop their communication and research skills respectively.